Technical Field of the Invention

The present invention relates to measurement of neural activity. In particular the invention relates to a method of measuring neural activity using a compound labelled with a detectable label which can target activated neural cells. The method finds use in the diagnosis o conditions associated with disturbed neural signalling.

Description of Related Art

Voltage-gated sodium channels (VGSCs) are essential for the initiation and propagation of neuronal impulses (Hodgkin and Huxley 1952 J Physiol; 1 17 (4): 500-44).

Specifically, these channels initiate the rising phase of action potentials in electrically excitable cells, allowing the conduction of electrical information. There are ten subtypes of VGSCs (Na 1.1 - 1 .9 and Na _v x), classification of which has allowed selective ligands to be designed and synthesised (Kyle and Ilyin 2007 J Med Chem; 50 (1 1 ): 2583-2588; Catterall 2000 Neuron; 26 (1 ); 13-25). VGSCs have been the subject of intense research due to their therapeutic potential for sufferers o conditions including epilepsy, neuropathic pain and cardiac arrhythmias (Nassar et al 2004 Proc Nat Acad Sci U S A; 101 (34): 12706-1271 1 ; Laird et al 2002 J Neurosci; 22 ( 19): 8352-6:

Mohler et al 2004 Proc Nat Acad Sci U S A; 101 (50): 17533-17538).

Recently, it has been reported that certain sub-types of VGSCs have been found in highly metastatic tumours (Fiske et al 2006 Can Met Rev; 25 (3): 493-500;

Brackenbury and Djamgoz 2006 J Physiol; 573 (2): 343-356; Roger et al 201 1 Oncog; 30 (17): 2070-2076). Moreover, the expression levels of VGSCs in these tumours are markedly increased when compared to naive tissue. Oncology research has shown their presence plays a key role in malignant progression and tumour invasiveness through up- regulation of key biochemical processes (Roger et al 2011 Oncog; 30 (17): 2070-2076).

Yang et al (2004 J Med Chem; 47 (6): 1547-1552) have reported 3-(4-phenoxyphenyl) pyrazoles that are state-dependent VGSC inhibitors with high in vitro potencies. 3-(4- phenoxyphenyl) pyrazole- 1 -carboxamides exhibited the greatest potencies with average inhibition constants (K,) for VGSCs in the inactivated state of 29 nM compared with 14.3 μΜ for the resting state. The structure of these most potent compounds is as follows:

wherein R is either para-F, para-NQi or ortho,para-F ₂ .

WO 2008/152109 disclosed labelled compounds based on the above compounds of Yang for use in imaging neural activity, including ^{i 8} F-labelled compounds of the above structure wherein R represents two substituents, one being F, and the other being a non-radioactive substituent selected from fluorine, N(¾, and bromine. In the experimental examples of WO 2008/1 52109 it was shown that in an animal

biodistribution model, a tritiated version of 3 -[2,4-Difluoro-phenoxy] -phenyl -pyrazole- 1 -carboxylic acid amide had good brain uptake and the time activity curves were consistent with reversible binding and the probe delineated grey and white matter (expected to have different levels of VGSCs). As concluded by Yang et al (supra), the amide functionality on the pyrazole group of these compounds is believed to be important for their affinity to the target. However, the present inventors have demonstrated with ¹⁸ F radiolabelling studies that this amide functionality is easily cleaved under basic radiolabelling conditions and consequently that these compounds may not be ideal as F-labelled PET tracer candidates. The present invention seeks to find ^{l x} F-labelled compounds that overcome the problems of the prior art and that are more suitable as PET imaging agents for VGSCs.

Summary of the Invention

The present invention provides a novel radiotluorinated compound for imaging VGSCs that is more straightforward to synthesise than the known radiotluorinated

phenoxyphenyl pyrazole carboxylic acid compounds. The compound of the present invention demonstrates specific uptake and retention in key tissues as well as good in vivo stability. Also provided by the present invention is a radiopharmaceutical composition comprising the radiofluorinated compound of the invention, precursor compounds and methods for the synthesis of said radiofluorinated compound, and methods for using said radiofluorinated compound.

Detailed Description of the Preferred Embodiments

In one aspect the present invention provides a radiofluorinated compound of Formula I:

wherein: each of R ¹ to R ³ is hydrogen, Ci _-3 alkyl, Ci _-3 alkoxy, thiol, Ci _-3 thioalkyl, Ci _-3 thioalkoxy, halo, C _1-3 haloalkyl, Ci„ ₃ haloalkoxy, nitro, nitroalkyl, or nitroalkoxy; and,

L ¹ represents a linker of formula -(A),,- wherein each A is independently -CR ₂ - , - CR=CR- , -C≡C- , -CR ₂ CO ₂ - , -CO ₂ CR2- , -NRCO- , -CONR- , -CR=N-0-, - NR(C=0)NR-, -NR(C=S)NR-, -S0 ₂ NR- , -NRS0 ₂ - , -CR ₂ OCR ₂ - , -CR ₂ SCR ₂ - , - CR2NRCR2- , a C4-8 cycloheteroalkylene group, a C _4-8 cycloalkylene group, -Ar-, - NR-Ar-, -O-Ar-, -Ar-(CO)-, an amino acid, a sugar or a monodisperse polyethyleneglycol (PEG) building block, wherein each Ar is independently a C5. ₁₂ arylene group, or a C _3- i ₂ heteroarylene group, and wherein each R is independently chosen from H, C ₁ -4 alkyl, C ₂ -4 alkenyl, C _2-4 alkynyl, C ₁ -4 alkoxy or C _1-4 hydroxyalkyl.

The term "radiofluorinated" means that the compound comprises a radioactive fluorine atom, i.e. ¹ F.

A suitable "salt" according to the invention includes (i) physiologically acceptable acid addition salts such as those derived from mineral acids, for example hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and those derived from organic acids, for example tartaric, trilTuoroacetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, methanesulphonic, and para-toluenesulphonic acids; and (ii) physiologically acceptable base salts such as ammonium salts, alkali metal salts (for example those of sodium and potassium), alkaline earth metal salts (for example those of calcium and magnesium), salts with organic bases such as

triethanolamine, N-methyl-D-glucamine, piperidine, pyridine, piperazine, and morpholine, and salts with amino acids such as arginine and lysine.

A suitable "solvate" according to the invention includes those formed with ethanol, water, saline, physiological buffer and glycol. The term "alkyl" used either alone or as part of another group is defined as any straight, branched or cyclic, saturated or unsaturated CJHbn ₊ i group. Examples of such radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec- butyl, tert-butyl, pentyl, iso-amyl, hexyl, octyl. Unless otherwise specified, the term "n" in the formula C _n H _2n+! is preferably between 1-6, most preferably between 1 -4. Unless otherwise specified, the term "alkoxy", alone or in combination, means an alkyl ether radical wherein the term alkyl is as defined above. Examples of suitable alkyl ether radicals include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert- butoxy.

The term "thiol" refers to the group -SH. The terms "thioalkvl" and "thioalkoxv" refer respectively to alkyl and alkoxy groups, respectively, as defined above substituted with one or more thiol groups.

The term "halo" means a substituent selected from fluorine, chlorine, bromine or iodine. "Haloalkyl" and "haloalkoxy" are alkyl and alkoxy groups, respectively, as defined above substituted with one or more halo groups. The term "nitro" refers to the group N0 ₂ . The terms "nitroalkvl" and "nitroalkoxv" refer respectively to alkyl and alkoxy groups, respectively, as defined above substituted with one or more nitro groups.

The term "cycloalkylene" refers to a cyclic alkylene. The term "alkylene" used either alone or in combination, refers to a straight or branched chai or cyclic bivalent aliphatic radical having a specified number of carbon atoms. Examples of alkylenes as used herein include, but are not limited to, methylene, ethylene, propylene, butyl ene and the like. The term "cycloheteroalkylene" refers to a cycloalkylene as defined above comprising a heteroatom selected from N, S or O.

The term "amino acid" refers to a bivalent group having the formula - HN- CH(R*)COO- wherien R* represents an organic substitutent. The term is intended to include L- and D-amino acids, amino acid analogues (e.g. naphthylalanine), which may be naturally occurring or of purely synthetic origin, and may be optically pure, i.e. a single enantiomer and hence chiral, or a mixture of enantiomers. Preferably the amino acids of the present invention are optically pure.

By the term "sugar" is meant a mono-, di- or tri- saccharide. Suitable sugars include: glucose, galactose, maltose, mannose, and lactose. The term "arylene". used either alone or in combination, refers to a bivalent unsaturated aromatic carboxylic radical having a single ring, such as phenyl ene, or multiple condensed rings, such as naphthylene or anthrylene. Examples of arylenes as used herein include, but are not limited to, benzene- 1 ,2-diyl, benzene- 1 , 3 -diyl, benzene- 1 ,4- diyl, naphthalene- 1 ,8-diyl, and the like. The term "heteroarylene" refers to an arylene as defined above comprising a heteroatom selected from N, S or O.

The term "alkynyl" as used herein refers to a bivalent moiety that contains at least one carbon-carbon triple bond and includes straight-chain, branched-chain and cyclic groups. The term "hydroxvalkvl" refers to an alkyl as defined herein substituted with a hydroxy group -OH.

The term "polvethvleneglycol" or "PEG" has its conventional meaning, as described e.g. in "The Merck Index", 14 ^th Edition entry 7568, i.e. a liquid or solid polymer of general formula H(OCH ₂ CH ₂ ),„OH where m is an integer greater than or equal to 4. The polyethyleneglycol polymers of the present invention may be linear or branched, but are preferably linear. Each of R ¹ to R ³ is preferably hydrogen, methyl, methoxy, thiol, thiomethyl, thiomethoxy, halo, halomethyl, halomethoxy, nitro, nitromethyl, or nitromethoxy, and most preferably hydrogen, methyl, methoxy, thiol, halo or nitro, and especially preferably hydrogen or halo.

R ¹ and R ² are particularly preferably both halo. R ³ is particularly preferably hydrogen.

Where one or more of R ¹ to R ³ is halo it is preferably fluoro, chloro or bromo, and most preferably fluoro. n is preferably an integer of 1 to 4, most preferably of 1 to 3, and especially preferably is 2. Each A in L ¹ is independently preferably CR ₂ or a monodisperse polyethyleneglycol (PEG) building block, most preferably -CR ₂ -, and most especially preferably -CH ₂ -.

In one preferred embodiment, R ¹ and R ² are at the 3- and 5-positions, respectively. In a more preferred embodiment, R ^l and R ² are at the 2- and 4-positions, respectively.

For a preferred compound of Formula I: each of R ¹ to R ³ is hydrogen, methyl, methoxy, thiol, thiomethyl, thiomethoxy, halo, halomethyl, halomethoxy, nitro, nitromethyl, or nitromethoxy; each A in L ¹ is independently CR ₂ or a monodisperse polyethyleneglycol (PEG) building block; and, n is an integer of 1 to 4.

For a most preferred compound of Formula I: each of R ¹ to R ³ is most preferably hydrogen, methyl, methoxy, thiol, halo or nitro; each A in L ¹ is CR ₂ ; and, n is an integer of 1 to 3.

For an especially preferred compound of Formula I: each of R ¹ to R ³ hydrogen or halo; each A in L ¹ is CH ₂ ; and, n is 2.

For each of the above-defined preferred compounds of Formula I, in a most preferred embodiment A is -CR ₂ - and most preferably -CH ₂ -. For a preferred radiofluorinated compound of the present invention ([ ^l8 F]9) excellent initial uptake in the brain (8.1 %id/g,) & heart (7.8 %id/g) was demonstrated at 2 minutes and clearance of the compound was seen within 2 hours. These results suggest specific uptake and retention of the compound in key tissues. In addition, the compound showed good in vivo stability. These results are set out in Example 5 below. The non-radioactive version of this preferred compound of the invention was compared with a prior art compound, as set out in Examples 6 and 7 below. Interestingly, a different onset of action was observed for 9 at 20 μΜ, than was seen with the prior art compound at 10 μΜ. 9 appeared to act like a traditional pore blocker; blocking action potentials without causing an effect on the spike amplitude. This is reminiscent of the site 1 blocker tetrodotoxin (TTX). The prior art compound, in contrast, showed use- dependent characteristics, having a greater onset of action upon longer stimulation. 9 is therefore believed to be a novel inhibitor of neuronal firing exhibiting TTX

characteristics.

In another aspect of the present invention is provided a radiopharmaceutical composition comprising the radiofluorinated compound as suitably and preferably defined herein together with a biocompatible carrier in a form suitable for mammalian administration.

The term "radiopharmaceutical composition" refers to a formulation comprising a radiolabelled compound or a salt thereof in a form suitable for administration to humans. Such a radiopharmaceutical composition may optionally contain further ingredients such as buffers; pharmaceutically acceptable solubilisers (e.g. cyclodextrins or surfactants such as Pluronic, Tween or phospholipids); pharmaceutically acceptable stabilisers or antioxidants (such as ascorbic acid, gentisic acid or para-aminobenzoic acid).

The "biocompatible carrier" is a fluid, especially a liquid, in which the radiotluorinated compound of the invention is suspended or dissolved, such that the composition is physiologically tolerable, i.e. can be administered to the mammalian body without toxicity or undue discomfort. The biocompatible carrier medium is suitably an injectable carrier liquid such as sterile, pyrogen-free water for injection; an aqueous solution such as saline (which may advantageously be balanced so that the final product for injection is either isotonic or not hypotonic); an aqueous solution of one or more tonicity- adjusting substances (e.g. salts of plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose), sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g. glycerol), or other non-ionic polyol materials (e.g. polyethyleneglycols, propylene glycols and the like). The biocompatible carrier medium may also comprise biocompatible organic solvents such as ethanol. Such organic solvents are useful to solubilise more lipophilic compounds or formulations. Preferably the biocompatible carrier medium is pyrogen-free water for injection, isotonic saline or an aqueous ethanol solution. The pH of the biocompatible carrier medium for intravenous injection is suitably in the range 4.0 to 10.5. The radi oph armaceuti cal composition of the invention may contain additional optional excipients such as: an antimicrobial preservative, pH-adjusting agent, filler,

radioprotectant, solubiliser or osmolality adjusting agent.

By the term "radioprotectant" is meant a compound which inhibits degradation reactions, such as redox processes, by trapping highly-reactive free radicals, such as oxygen-containing free radicals arising from the radiolysis of water. The

radioprotectants of the present invention are suitably chosen from: ethanol, ascorbic acid, /? ra-aminobenzoic acid (i.e. 4-aminobenzoic acid), gentisic acid (i.e. 2,5- dihydroxybenzoic acid) and salts thereof with a biocompatible cation. By the term "biocompatible cation" (B ^c ) is meant a positively charged counterion which forms a salt with an ionised, negatively charged group, where said positively charged counterion is also non-toxic and hence suitable for administration to the mammalian body, especially the human body. Examples of suitable biocompatible cations include: the alkali metals sodium or potassium; the alkaline earth metals calcium and magnesium; and the ammonium ion. Preferred biocompatible cations are sodium and potassium, most preferably sodium.

By the term "solubiliser" is meant an additive present in the radiopharmaceutical composition which increases the solubility of the imaging agent in the solvent. A preferred such solvent is aqueous media, and hence the solubiliser preferably improves solubility in water. Suitable such solubilisers include: C alcohols; glycerine;

polyethylene glycol (PEG); propylene glycol; polyoxyethylene sorbitan monooieate; sorbitan monooloeate; polysorbates;

poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block copolymers

(Pluronics™); cyclodextrins (e.g. alpha, beta or gamma cyclodextrin, hydro xypropyl- β - cyclodextrin or hydroxypropyl-y-cyclodextrin) and lecithin.

By the term "antimicrobial preservative" is meant an agent which inhibits the growth of potentially harmful micro-organisms such as bacteria, yeasts or moulds. The antimicrobial preservative may also exhibit some bactericidal properties, depending on the dosage employed. The main role of the antimicrobial preservative(s) of the present invention is to inhibit the growth of any such micro-organism in the

radiopharm aceuti cal composition. The antimicrobial preservative may, however, also optionally be used to inhibit the growth of potentially harmful micro-organisms in one or more components of kits used to prepare said composition prior to administration. Suitable antimicrobial preservative(s) include: the parabens, i.e. methyl, ethyl, propyl or butyl paraben or mixtures thereof; benzyl alcohol; phenol; cresol; cctrimide and thiomersal. Preferred antimicrobial preservative(s) are the parabens.

The term "pH-adjusting agent" means a compound or mixture of compounds useful to ensure that the pH of the radiopharmaceutical composition is within acceptable limits (approximately pH 4.0 to 10.5) for human or mammalian administration. Suitable such pH-adjusting agents include pharmaceutically acceptable buffers, such as tricine, phosphate or TRIS [i.e. tm(hydroxymethyl)aminomethane], and pharmaceutically acceptable bases such as sodium carbonate, sodium bicarbonate or mixtures thereof. When the radiopharmaceutical composition is employed in kit form, the pH adjusting agent may optionally be provided in a separate vial or container, so that the user of the kit can adjust the pH as part of a multi-step procedure.

By the term "filler" is meant a pharmaceutically acceptable bulking agent which may facilitate material handling during production and lyophilisation. Suitable fillers include inorganic salts such as sodium chloride, and water soluble sugars or sugar alcohols such as sucrose, maltose, mannitol or trehalose. The radiofluorinated compounds of the present invention may be prepared either from an alkyne precursor compound of Formula II as described below, or from a nonradioactive derivative comprising a leaving group of Formula III as described thereafter.

A further aspect of the present invention is a precursor compound of Formula II:

wherein R to R are as suitably and preferably defined herein for R to R , respectively; which upon reaction with N ₃ -L - F results in the radiofluorinated compound of the present invention, wherein L ² is as suitably and preferably defined herein for L ¹ .

A "precursor compound" comprises a derivative of a labelled compound, designed so that chemical reaction with a convenient chemical form of a label occurs site-specifically; can be conducted in the minimum number of steps (ideally a single step); and without the need for significant purification (ideally no further purification), to give the desired labelled compound. Such precursor compounds are synthetic and can conveniently be obtained in good chemical purity. The precursor compound may optionally comprise a protecting group for certain functional groups of the precursor compound. Protecting groups are described in 'Protective Groups in Organic Synthesis', Theorodora W. Greene and Peter G. M. Wuts, (Fourth Edition, John Wiley & Sons, 2006).

The term "suitably and preferably defined" as used here and elsewhere in the specification is intended to encompass those embodiments that are essential and preferred, including in addition those embodiments that are cited as e.g. most preferred, especially preferred, and most especially preferred.

Scheme 1 below illustrates the synthetic route for obtaining precursor compounds of Formula II of the present invention (wherein R ⁴ to R ⁶ are as suitably and preferably defined herein for Formula II):

The commercially-available phenol and ethynyl fluorobenzene starting compounds are reacted under Williamson reaction conditions to produce the precursor compound of Formula II. R ⁴ to R ⁶ are as suitably and preferably defined herein.

A yet further aspect of the invention is a precursor compound of Formula III:

wherein R ⁷ to R ⁹ are as suitably and preferably defined herein for R ^! to R ³ , respectively; L ² is as suitably and preferably defined for L ¹ herein; and,

LG is a leaving group; which upon reaction with ^{, 8} F-fluoride results in the radiofluorinated compound of the present invention. The term "leaving group" used here and elsewhere in the specification refers to a molecular fragment that departs with a pair of electrons in heterolytic bond cleavage. A suitable leaving group can be a halo, e.g. selected from chloro, iodo, or bromo, or an aryl or alkyl sulphonate, for example, tosylate, triflate, nosylate or mesylate.

The precursor compound of Formula III may be obtained starting with the method to obtain said precursor compound of Formula II followed by reaction with N ₃ -L ³ -LG as illustrated in the following reaction scheme (wherein R ⁴ to R ⁶ , L ³ and LG are as suitably and preferably defined herein for Formula III):

It is well-known in the art how to obtain a wide variety of azide compounds (Organic Azides: Synthesis and Applications 2010; Wiley: Brase and Banert, Eds). These methods can be applied to obtaining the azide N ₃ -L ³ -LG in the above synthetic scheme for obtaining precursor compounds of Formula III. Demko and Sharpless (2001 Org Lett; 3(25): 4091- 4094) describe preparation of azides by conversion of the corresponding bromo-alcohol of formula Br-(CH ₂ ) _n -OH to the corresponding azido-alcohol N ₃ -(CH ₂ ) _n -OH, followed by conversion to the tosylate with toluenesulfonyl chloride in the presence of triethylamine. An alternative method is S- _N -2 displacement with azide of a ditosylate species as detailed below (reaction 1 ). A further method is described for PEGylated chains in Svedhem et al, (2001 J Org Chem: 4494) (reaction 2): N ₃ Reaction 1

Reaction 2

In the above scheme, DCM is dichloromethane and MsCl is methanesulfonyl chloride.

The reaction illustrated above to obtain precursor compounds of Formula III is carried out in the presence of a click catalyst. By the term "click catalyst" is meant a catalyst known to catalyse the click (i.e. alkyne plus azide) reaction. Suitable such catalysts are known in the art for use in click reactions. A preferred click catalyst comprises Cu(I). The Cu(I) catalyst is present in an amount sufficient for the reaction to progress, typically either in a catalytic amount or in excess, such as 0.02 to 1 .5 molar equivalents relative to the azide. Suitable Cu(I) catalysts include Cu(l) salts such as Cul or [Cu(NCCH ₃ ) ₄ ][PF ₆ ], but advantageously Cu(Il) salts such as copper (II) sulfate may be used in the presence of a reducing agent to generate Cu(I) in situ. Suitable reducing agents include: ascorbic acid or a salt thereof for example sodium ascorbate, hydroquinone, metallic copper, glutathione, cysteine, Fe ²⁺ , or Co ²⁺ . Cu(I) is also intrinsically present on the surface of elemental copper particles, thus elemental copper, for example in the form of powder or granules may also be used as catalyst. Elemental copper, with a controlled particle size is a preferred source of the Cu(I) catalyst. A more preferred such catalyst is elemental copper as copper powder, having a particle size in the range 0.001 to 1 mm, preferably 0.1 mm to 0.7 mm, more preferably around 0.4 mm. Alternatively, coiled copper wire can be used with a diameter in the range of 0.01 to 1.0 mm, preferably 0.05 to 0.5 mm, and more preferably with a diameter of 0.1 mm. The Cu(I) catalyst may optionally be used in the presence of bathophenanthroline, which is used to stabilise Cu(I) in click chemistry.

Further details of suitable catalysts are described by Wu and Fokin (2007 Aldrichim Acta; 40(1 ): 7- 17) and by Meldal and Tornoe (2008 Cfaem Rev; 108: 2952-3015).

The click reaction may be carried out in a suitable solvent, for example acetonitrile, a C alkylalcohol, dimethylformamide, tetrahydrofuran, or dimethylsulfoxide, or aqueous mixtures of any thereof, or in water. Aqueous buffers can be used in the pH range of 4-8, more preferably 5-7. The reaction temperature is preferably 5 to 100°C, more preferably 75 to 85°C, most preferably ambient temperature (typically 1 5-37 °C). The click reaction may optionally be carried out in the presence of an organic base, as is described by Meldal and Tornoe, supra.

In another further aspect of the present invention is provided a method for the synthesis of the radiofluorinated compound as suitably and preferably defined herein wherein said method comprises:

(i) providing the precursor compound of Formula II as suitably and preferably defined herein;

(ii) reacting said precursor with N ₃ -L ² - ¹⁸ F in the presence of a click catalyst to obtain said radiofluorinated compound.

The features and conditions described above for the synthesis of the precursor compound of Formula III are equally applicable for this synthesis of the radiofluorinated compound of Formula I of the present invention.

N ₃ -L - F for this aspect of the invention is as suitably and preferably defined above. This reactant is obtained using the methods described above for the synthesis of N ₃ -L ³ -LG, followed by reaction with [ ^!8 F] fluoride.

In another aspect, the present invention also provides a method for the synthesis of the radiofluorinated compound as suitably and preferably defined herein wherein said method comprises: (i) providing the precursor compound of Formula III as suitably and preferably defined herein;

(ii) reacting said precursor with ¹⁸ F-fiouride to obtain said radiofluorinated compound.

[ ¹⁸ F]-fluoride for use in the above-described radioiluorination reactions is normally obtained as an aqueous solution from the nuclear reaction ^{! 8} 0(p,n) ^!8 F. In order to increase the reactivity of fluoride and to avoid hydroxylated by-products resulting from the presence of water, water is typically removed from [ ¹⁸ F] -fluoride prior to the reaction, and fluorination reactions are carried out using anhydrous reaction solvents (Aigbirhio et al 1995 J Fluor Chem; 70: 279-87). The removal of water from [ ^{! 8} F] -fluoride is referred to as making "naked" [ ^l8 F] -fluoride. A further step that is used to improve the reactivity of

[ F] -fluoride for radioiluorination reactions is to add a cationic counterion prior to the removal of water. Suitably, the counterion should possess sufficient solubility within the anhydrous reaction solvent to maintain the solubility of the [ ⁱ⁸ F] -fluoride. Therefore, counterions that are typically used include large but soft metal ions such as rubidium or caesium, potassium complexed with a cryptand such as Kryptofix , or tetraalkylammonium salts, wherein potassium complexed with a cryptand such as Kryptofix™, or tetraalkylammonium salts are preferred.

Both of the above-described methods for the synthesis of the radiofluorinated compound of Formula 1 are preferably carried out in an aseptic manner, such that the radiofluorinated compound of Formula I is obtained as the radiopharmaceutical composition of the invention. It is preferred therefore that the key components, especially any parts of the apparatus which come into contact with the reactants and product, e.g. vials and transfer tubing, are sterile. The components and reagents can be sterilised by methods known in the art, including: sterile filtration, terminal sterilisation using e.g. gamma-irradiation, autoclaving, dry heat or chemical treatment (e.g. with ethylene oxide). It is preferred to sterilise the non-radioactive components in advance, so that the minimum number of manipulations need to be carried out on the radiopharmaceutical product. As a precaution, however, it is preferred to include at least a final sterile filtration step.

The reactants are suitably supplied in vials or vessels which comprise a sealed container which permits maintenance of sterile integrity and/or radioactive safety, plus optionally an inert headspace gas, e.g. nitrogen or argon, whilst permitting addition and

withdrawal of solutions by syringe or cannula. A preferred such container is a septum- sealed vial, wherein the gas-tight closure is crimped on with an overseal (typically of aluminium). The closure is suitable for single or multiple puncturing with a

hypodermic needle (e.g. a crimped-on septum seal closure) whilst maintaining sterile integrity. Such containers have the additional advantage that the closure can withstand vacuum if desired (e.g. to change the headspace gas or degas solutions), and withstand pressure changes such as reductions in pressure without permitting ingress of external atmospheric gases, such as oxygen or water vapour. The reaction vessel is suitably chosen from such containers, and preferred embodiments thereof. The reaction vessel is preferably made of a biocompatible plastic (e.g. PEEK). In addition, both of the above-described methods for the synthesis of the radiofluorinated compound of Formula I are preferably carried out using an automated synthesiser apparatus. By the term "automated synthesiser" is meant an automated module based on the principle of unit operations as described by Satyamurthy et al (1999 Clin Positr Imag; 2(5): 233-253). The term 'unit operations' means that complex processes are reduced to a series of simple operations or reactions, which can be applied to a range of materials. Such automated synthesisers are preferred for the method of the present invention especially when the radiopharmaceutical composition of the invention is desired. They are commercially available from a range of suppliers (Satyamurthy et al, supra), including: GE Healthcare; CTl Inc; Ion Beam Applications S.A.(Chemin du Cyclotron 3, B-1348 Louvain-La-Neuve, Belgium); Raytest (Germany) and Bioscan (USA).

Commercial automated synthesisers also provide suitable containers for the liquid radioactive waste generated as a result of the radiopharmaceutical preparation. Automated synthesisers are not typically provided with radiation shielding, since they are designed to be employed in a suitably configured radioactive work cell. The radioactive work cell provides suitable radiation shielding to protect the operator from potential radiation dose, as well as ventilation to remove chemical and/or radioactive vapours. Preferred automated synthesisers of the present invention are those which comprise a disposable or single use cassette which comprises all the reagents, reaction vessels and apparatus necessary to carry out the preparation of a given batch of radiopharmaceutical. Such cassettes represent additional aspects of the invention described below. The cassette means that the automated synthesiser has the flexibility to be capable of making a variety of different radiopharmaceuticals with minimal risk of cross-contamination, by simply changing the cassette. The cassette approach also has the advantages of: simplified set-up hence reduced risk of operator error; improved GMP (Good Manufacturing Practice) compliance; multi- tracer capability; rapid change between production runs; pre-run automated diagnostic checking of the cassette and reagents; automated barcode cross-check of chemical reagents vs the synthesis to be carried out; reagent traceability; single-use and hence no risk of cross- contamination, tamper and abuse resistance.

In an additional aspect the present invention provides a cassette for the automated synthesis of the radiofluorinated compound of Formula I as suitably and preferably defined herein wherein said cassette comprises:

(i) a vessel containing a precursor compound o Formula II as suitably and preferably defined herein; and,

(ii) means for eluting the vessel with N ₃ -L ² - ¹⁸ F as suitably and preferably defined herein. In another additional aspect the present invention provides cassette for the automated synthesis of the radiofluorinated compound of Formula I as suitably and preferably defined herein wherein said cassette comprises:

(i) a vessel containing a precursor compound of Formula III as suitably and preferably defined herein; and, (ii) means for eluting the vessel with ¹⁸ F-fluoride.

The radiofluorinated compound of Formula I of the present invention finds use in determining the expression of voltage-gated sodium channels (VGSC) in a subject. The present invention therefore provides in another aspect an in vivo imaging method to determine the location and/or quantity in a subject of VGSC wherein said method comprises detection of signals emitted by the radiofluorinated compound of the in vention present in said subject.

The in vivo imaging method of the present invention comprises: (i) administering the radiofluorinated compound as suitably and preferably defined herein to said subject;

(ii) allowing said radiofluorinated compound to bind to VGSC in said subject;

(iii) detecting signals emitted by the ^{, 8} F present on said bound radiofluorinated compound; and,

(iv) converting said signals into an image representative of the location and/or quantity in said subject of VGSC.

The in vivo imaging method of the invention is suitably positron emission tomography (PET). PET affords images by virtue of the detection of pairs of gamma rays emitted indirectly by a positron-emitting radiotracer. Images of tracer concentration within the body are constructed by computer analysis. In modern scanners, 3D imaging is often accomplished with the aid of a computed tomography (CT) scan performed concurrently. In general, PET/CT imaging offers the ability to non-invasively and quantitatively diagnose diseases to allow appropriate treatment selection.

The "subject" for this aspect of the invention is preferably a mammalian subject, most preferably a human subject.

The radiofluorinated compound is preferably administered intravenously. For the in vivo imaging method of the invention it is preferred that the radiofluorinated compound is administered as said radiopharmaceutical composition of the present invention.

The in vivo imaging method of the invention may optionally be carried out repeatedly to monitor the effect of treatment of said subject with a drug, said in vivo imaging being effected before and after treatment with said drug, and optionally also during treatment with said drug. Step (i) of the in vivo imaging method of the present invention can alternatively be a step of providing said subject wherein said radiofluorinated compound has been pre-administered to said subject. The term "pre-administered" refers to wherein the radiofluorinated compound has been administered to the patient prior to the in vivo imaging method of the invention.

The in vivo imaging method of the invention may be applied to evaluate normal physiology, or to determine the presence of a condition associated with altered expression of VGSC. Examples of such conditions include (but are not necessarily limited to) neurological conditions associated with disturbed neural signalling such as epilepsy, pathological pain, multiple sclerosis, Parkinson's disease, Alzheimers, schizophrenia, and depression; carcinomas such as prostate cancer, breast cancer and small cell lung cancer; and chronic pain. In a further aspect therefore the present invention provides a method to diagnose in a subject such conditions associated with altered expression ofVGSC, wherein said method comprises the in vivo imaging method as suitably and preferably defined above in addition to the subsequent steps:

(v) comparing an image obtained in accordance with the method as suitably and preferably defined herein for said subject with standardised images obtained from healthy volunteers;

(vi) finding any significant deviation between the image for said subject and the standardised images from healthy volunteers; and,

(vii) attributing said deviation to a particular clinical picture.

In the alternative, the present invention also comprises the radiofluorinated compound of Formula I as suitably and preferably defined herein for use in either the in vivo method of the present invention or the method to diagnose of the present invention.

In a further alternative, the present invention comprises use of the radiofluorinated compound of Formula I as suitably and preferably defined herein in the manufacture of the rad i opharmaceuti cal composition as suitably and preferably defined herein for use in either the in vivo method of the present invention or the method to diagnose of the present invention.

The present invention is now described in terms of the following non-limiting examples.

Brief Description of the Examples

Comparative Example 1 describes the attempted F-alkylation of a 3-(4- phenoxyphenyl) pyrazole- 1 -carboxamide. Comparative Example 2 describes the biological evaluation of 3-(4-(2,4- difluorophenoxy)phenyl)- 1 -(2-fluoroethyl)- 1 H-pyrazole (4).

Example 3 describes the synthesis of 4-(4-(2,4-difluorophenoxy)phenyl)- 1 -(2- fluoroethyl)- 1 H- 1 ,2,3-triazole (9).

Example 4 describes the synthesis of 4-(4-(2,4-difiuorophenoxy)phenyl)-l-(2- [ 18F]fluoroethyl)- 1 H- 1 ,2,3-triazole (| ^W F]9).

Example 5 describes the biological evaluation of [ ^l F]9.

Comparative Example 6 describes the electrophysiological evaluation of the prior art compound 3-(4-(2,4-difluorophenoxy)phenyl)- 1 H-pyrazole- 1 -carboxamide.

Example 7 describes the electrophysiological evaluation of [ ^,8 F]9. Example 8 describes a metabolism study of [ ¹⁸ FJ9.

List of Abbreviations used in the Examples

AHPs after-hyperpolarisations

CA1 cornu ammonis 1 (one of 4 histological divisions of the hippocampus)

DMAP dimethylaminopyridine

DMF dimethylform amide

EOS end of synthesis

EtOH ethanol HPLC high performance liquid chromatography hr(s) hour(s) id/g injected dose per gram min(s) minute(s) n.d.c non-decay corrected

PC piriform cortex rt room temperature

VGSC(s) voltage-gated sodium channel(s) v/v volume to volume Examples

Commfat Exa.m≠e I: Attempted "F-aikflatimt of a 3~{4~phmMxyphm≠)

pvrazole-l-carboxam ide

Figure 1 illustrates the synthetic scheme. 3-(4-(2,4-difluorophenoxy)phenyl)- 1 H- pyrazole- 1 -carboxamide 1 was reacted with [ ¹⁸ F]fluoroethyltosylate ([ ^l8 F|2)with the aim that alkylation would proceed on the primary carboxamide. However, total cleavage of the carboxamide was seen using a range of bases, even mild sodium bicarbonate. It was found that alkylation on the pyrazole nitrogen proceeded in excellent yield using sodium hydride as a base giving [ ¹⁸ F]4 as the major isomer in 1 1 % end of synthesis (EOS) n.d.c. yield from fluoride in 90-95% radiochemical purity. Figure 2 shows the analytical HPLC trace of the major isomer [ ^!8 F]4 and the minor isomer

[ ¹⁸ F|5, eluting at 8.01 minutes and 7.40 minutes, respectively.

Comparative Example 2: Biological Evaluation of 3-(4-(2,4- difluorophenoxv)phenvl)-l-(2-fluoroethvl)-lll-pvrazole (4) [ ¹⁸ F]4 was assessed through naive mice edistribution studies. [ ⁱ⁸ F]4 was delivered in (9:1 v/v) phosphate buffer /ethanol solution at 30.3 MBq per ml. The radiotracer was sampled at five time points over 2 hrs three animals were sacrificed at each time point.

The biodistribution (illustrated in Figure 3) showed good initial brain (2 mins, 6.3 %id/g) and heart (2 mins, 5.9 %id/g) uptake but within 30 mins it had cleared to brain ( 1 .5 %id/g) and heart (1 .8 %id/g). From 15 mins to 120 mins the data suggests nonspecific uptake and shows little retention of | ^1!i F]4 in key tissues. It was therefore concluded that the structural modifications had most likely decreased or removed the affinity for VGSCs. Example 3: Synthesis of ^^i ! ≠W iiBhS!S tLQ^^^M&. 1.2,3-triazole (9)

The synthesis is illustrated in Figure 4. Reaction of 2,4 difluorophenol with 1-ethynyl- 4-fluorobenzene under Williamson reaction conditions produced 6 in 15% yield. 8 was produced starting from 2-fluoroethanol converted to the tosylate 7 using tosyl chloride under basic conditions catalysed by dimethylaminopyridine (DMAP). Tosyl was replaced using sodium azide in N,N DMF to yield 8. Due to the instability of 8, it remains as a solution in NNDMF with concentration of 0.23 mmol/ml. The subsequent Huisgen 1 ,3 cycloaddition reaction between 8 and 6 uses copper wire (where copper (I) is present on the surface) as catalyst affording 9 in 1 0% yield. mmiiLiLSM

lH-l,2,3-triazole (l ^w F19)

Figure 5 illustrates the synthesis. [ ¹ F]8 was synthesised according to the method of Iddon et al (201 1 Bioorg Med Chcm Letts; 21 ( 10): 3 122-31 2). The precursor was produced in a two-step synthesis, bromoethanol was converted to 10 in the presence of sodium azide in water refluxed for 1 6 hrs via an S 2 reaction. Due to the volatile nature of 10 this compound was not isolated. Pyridine and tosyl chloride were added at 0 °C and the reaction proceeded at room temperature (rt) via another S 2 reaction to yield 11 m 90% yield. [ ^I8 F]8 was produced in 20-30% n.d.c. yield. Copper (I) was produced under the classic reduction of copper(II) sulphate with sodium ascorbate in water and the addition of the ligand bathophenanthroline. The cycloaddition consequently proceeded in 37% non-decay corrected yield giving [ ^,!t F]9 in 10% end of synthesis yield, n.d.c from fluoride. Example 5: Biolosical Evaluation ofi ^ls F19

[ ¹⁸ F]9 was characterised through naive mice biodistribution (illustrated in Figure 6). was delivered in (9: 1 v/v) phosphate buffer /ethanol solution at 25.0 MBq per ml. The radiotracer concentration was sampled at five time points over 2 hrs, three animals were sacrificed at each time point. [ ^,8 F]9 showed excellent initial uptake in the brain (8.1 %id/g, 2 mins) and heart (7.8 %id/g, 2 mins). After 30 mins there was still a high degree of binding in the brain (4.0 %id/g) and heart (5.1 %id/g). After 120 mins the tracer appears to have cleared.

.gf ifiggii^ ··**·' QmmmM

The prior art compound 3-(4-(2,4-difluorophenoxy)phenyl)-l H-pyrazole- l-carboxamide has been shown by Yang et at to have K, of 25 nM:

Brain slices were obtained from Sprague-Dawley rats. The brain was rapidly removed following decapitation, hemisected and placed in ice-cold oxygenated Kreb's solution at 4 °C. Transverse slices were cut 450 μιη thick using a vibrating tissue slicer in ice-cold Kreb's medium. Once the slices had been cut, they were transferred to a Kreb's solution warmed to 29 °C for 1 hour to recover. Recordings were taken from pyramidal cells within layers II- III of the piriform cortex (PC), using a 1 μΜ solution of the prior art compound (1 :99 EtOH/Kreb's). The piriform cortex (PC) was studied, due to good neuronal activity in this area and extensive characterisation reported in the literature. A pyramidal cell of the PC was impaled and held at a membrane potential of -70 mV. A pyramidal cell of the CA1 region was impaled and held at -70 mV. Control recordings were acquired before applying the prior art compound at 10 μΜ for 45 mins.

At 15 min intervals, recordings were taken to monitor the drug effect. At 45 mins the drug effect peaked. The number of action potentials and the peak spike amplitude after the first spike decreased dramatically. For example, the amplitude of the 4 ^th spike decreased by 12 mV from 65 mV in control to 53 mV in the prior art compound. After 45 min washout, the spike amplitude showed some recovery, returning to 60 mV. The time between spikes also increased due to the longer spike after-hyperpolarisations (AHPs), most likely due to opening of more calcium channels. The time between action potentials 4 and 5 was 20 ms in control, whereas after application of prior art compound at 10 μΜ for 45 mins, this gap doubled to 40 ms. After washout, this time did not change remaining at 40 ms, indicating the drug had not washed out after 45 mins. Meanwhile, the resting membrane potential remained steady and there was no change in slow inward rectification currents or input resistance. Having raised the drug concentration 10-fold, the prior art compound had now exhibited reversible binding to the pyramidal cells of the CA1 with some slow washout seen after 45 mins.

Example 7: Electroph ysiolosical Evaluation of 9

9 (i.e. non-radioactive [ ^,8 F]9) was tested using the model described in Comparative Example 6. A 20 μΜ solution of 9 (2:98 EtOH/Kreb's) bubbled with oxygen 95% / carbon dioxide 5% was applied to the system and neuronal activity was monitored.

Recordings were taken 20 mins after a 20 μΜ solution of 9 was applied. After 7 mins, a drop in the number of action potentials fired was observed. Only a very slight drop in the spike amplitude was seen (65 mV to 60 mV), whilst AHPs were again increasing due to opening of calcium channels. Within 20 mins, total block of all action potentials were seen. When applying the long pulse (1.5 sec) some action potentials were seen indicating reversible binding with longer stimulation. 9 has affinity for blocking neuronal firing in the pyramidal cells of the CA1 region at 20 μΜ. Example 8: Metabolism off ^,8 FI9

[ F|9 (synthesised as per Example 4) was delivered in 7.3% n.d.c. e.o.s. yield from fluoride at 104 MBq/mL as a phosphate buffer /ethanol solution (9: 1 ; v/v), with 0.187 specific activity GBq/μηιοΙ. 10.4 MBq (100 μΐ), and was injected via the tail vein to each C57BL/6 naive male mouse.

The radiotracer concentration was sampled at three time points (2, 15 and 60 minutes) in the heart and plasma and at 15 minutes only in the brain. One animal was sacrificed at each time point (except 60 minutes, where two animals were sacrificed and their tissue combined due to little activity remaining at later time points). Once the tissue was extracted at each time, the tissue samples were cleaned, homogenised and separated using the protein precipitation method starting with the addition of 5 ml, ice-cold acetonitrile and centrifuged for 3 minutes at 1000 rpm. The supernatant was removed and collated in a 100 mL round bottom flask. The total volume of supernatant was recorded and 100 μΐυ aliquoted into an eppendorf tube for measuring at the end of the study in duplicate. The pellet was placed onto a Wallach counter to measure the efficiency of the centrifugation step. The remaining supernatant was put on the rotary evaporator to remove the acetonitrile and resuspended in 2 mL (40% MeCN and 60% H ₂ 0) mobile phase. A 100 μΕ aliquot of the mobile phase solution was taken for counting in duplicate. Of the remaining mobile phase solution, 1 mL was injected onto a semi-preparative HPLC (gradient of 40-100% MeCN, 3 ml/min). The eluent was collected to ascertain the efficiency of the HPLC purification. This procedure was then repeated for each sample over the three time points.

The table below shows the percentage parent vs non-parent for each time point in the heart and plasma. A 15 minute brain sample was taken to see whether the uptake shown in the brain during the biodistribution was due to uptake of parent or non-parent:

Overall, the study showed that uptake in the heart and brain is mainly parent radiotracer (at 2 and 15 minutes) and not a metabolite. While after 60 minutes the amount of parent radiotracer decreases and more metabolite is seen, which is consistent with the biodistnbution data, as the signal in these tissues decreases after 60 minutes. This metabolism study indicates adequate in vivo stability of | ^,8 F]9.